Genome wide inherited modifications of the tomato epigenome by trans-activated bacterial CG methyltransferase

Background Epigenetic variation is mediated by epigenetic marks such as DNA methylation occurring in all cytosine contexts in plants. CG methylation plays a critical role in silencing transposable elements and regulating gene expression. The establishment of CG methylation occurs via the RNA-directed DNA methylation pathway and CG methylation maintenance relies on METHYLTRANSFERASE1, the homologue of the mammalian DNMT1. Purpose Here, we examined the capacity to stably alter the tomato genome methylome by a bacterial CG-specific M.SssI methyltransferase expressed through the LhG4/pOP transactivation system. Results Methylome analysis of M.SssI expressing plants revealed that their euchromatic genome regions are specifically hypermethylated in the CG context, and so are most of their genes. However, changes in gene expression were observed only with a set of genes exhibiting a greater susceptibility to CG hypermethylation near their transcription start site. Unlike gene rich genomic regions, our analysis revealed that heterochromatic regions are slightly hypomethylated at CGs only. Notably, some M.SssI-induced hypermethylation persisted even without the methylase or transgenes, indicating inheritable epigenetic modification. Conclusion Collectively our findings suggest that heterologous expression of M.SssI can create new inherited epigenetic variations and changes in the methylation profiles on a genome wide scale. This open avenues for the conception of epigenetic recombinant inbred line populations with the potential to unveil agriculturally valuable tomato epialleles. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05255-7.

F3 plants exhibiting the abnormal leaf phenotype and M82 used as control were pictured 30 days after germination.The phenotype consistently manifests in plants that overexpress M.SssI, although full penetrance is not achieved, as evidenced by the fact that not all plants carrying the activation system display the phenotype.Indeed, investigations into F3 progenies of an F2 plant double heterozygous for the two constructs and showing the leaf phenotype revealed that 10 out of 54 plants exhibited the phenotype.Conversely, in F3 progenies of an F2 plant lacking the M.SssI activation system, the phenotype was completely absent, with none of the 50 progeny plants showing it.Sup Figure 10 Sup Figure 10.Nature of the CG DMRs identified between the F2-Mss(+) plants and the driver line control plants."Gene+TE" are DMRs overlapping with both genes and transposons, "Gene", DMRs overlapping with genes, and "TE" DMRs overlapping with Transposable Elements (TEs).All other DMRs were classified as "Intergenic".

Sup Figure 2 .
Transient expression of M.SssI in Nicotiana benthamiana leaves results in cytosine methylation Quantification of global 5-methyl cytosine in the genomic DNA of Nicotiana benthamiana leaves two days post infiltration with pART27_disM.SssI and pART27_2X35S used as control.Error bars indicate ±SD over two biological replicates.Sup Figure 3. Abnormal leaf phenotype observed in tomato plants overexpressing disM.SssI.
Methylated cytosines (CG, CHG and CHH methylation contexts are plotted together) in the plants analysed.Reads were aligned to the three transgene components: the M.SssI gene, the pFIL promotor and the LhG4 gene.The methylation levels are given in % (scale: 0 to 100%).

5 .Sup Figure 6 .Sup Figure 7 .F2 8 . 9 .
Methylation along the 12 chromosomes of tomato, calculated from non-overlapping 200 kb bins.The average methylation levels were determined by combining the different plants for each genotype.Only cytosines covered by at least five reads were considered and only bins containing at least 10 valid cytosines were kept.Control: pFIL::LhG4 driver line.Correlation network diagram constructed using Spearman's correlation coefficients to illustrate the relationships among the CG methylation bins depicted in the boxplots of Figure3B.The lines represent the strength of the correlation: wider lines mean a stronger correlation.The spacing between the nodes is also indicative of the correlation level.Only correlations that are statistically significant (P-value < 0.05) are shown.cn, control driver line plants #1 and #2.Patterns of methylation in genes in the four Mss-F2(+) plants expressing disM.SssI (blue lines) and the control drive lines (orange lines).The average methylation level of genes was determined by dividing the corresponding annotated regions into 100 bp bins.Regions located 1 kb upstream and 1 kb downstream of genes are shown.The repeat-rich and repeat-poor regions were defined as previously described(Jouffroy et al., 2016), using the SL2.50 version of the genome assembly.Patterns of methylation in Transposable Elements (TEs) in the four Mss-F2(+) plants expressing disM.SssI (blue lines) and the control drive lines (orange lines).The average methylation level of TEs was determined by dividing the corresponding annotated regions into 100 bp bins.Regions located 1 kb upstream and 1 kb downstream of the TEs are shown.The repeat-rich and repeatpoor regions were defined as previously described(Jouffroy et al., 2016), using the SL2.50 version of the genome assembly.Number of DMRs detected in F2 and F3 plants compared to the control driver line plants.Repeat-Poor regions (RP), Repeat-Intermediate regions (INT), and Repeat-Rich regions (RR) were defined as previously described (Jouffroy et al., 2016), using the SL2.50 version of the genome assembly.

F2 11 . 12 . 13 .
Patterns of methylation for CG hyperDMRs identified between F2-Mss(+) plant#1 and driver line controls.Metaprofiles showing the methylation levels in the F2-Mss(+) transgenic lines expressing both the pFIL::LhG4 and pOP::disM.SssI transgenes (the four blue lines), the control driver line expressing only pFIL::LhG4 (the four red lines), the F2-Mss(-) plants carrying no transgenes (the two orange lines) and the F3-Mss(-) plants carrying only the pFIL:LhG4 transgene (the two green lines).The average methylation level of DMRs was determined by dividing the corresponding annotated regions into 100bp-bins.Regions located 2 kb upstream and 2 kb downstream of the CG hyperDMR are shown.Patterns of methylation for CG hyperDMRs identified between F2-Mss(+) plant#3 and driver line controls.Metaprofiles showing the methylation levels in the F2-Mss(+) transgenic lines expressing both the pFIL::LhG4 and pOP::disM.SssI transgenes (the four blue lines), the control driver line expressing only pFIL::LhG4 (the four red lines), the F2-Mss(-) plants carrying no transgenes (the two orange lines) and the F3-Mss(-) plants carrying only the pFIL:LhG4 transgene (the two green lines).The average methylation level of DMRs was determined by dividing the corresponding annotated regions into 100bp-bins.Regions located 2 kb upstream and 2 kb downstream of the CG hyperDMR are shown.Patterns of methylation for CG hyperDMRs identified between F3-Mss(-) and driver line control plants.Metaprofiles show the methylation levels in the four F2-Mss(+) transgenic lines expressing both pFIL::LhG4 and pOP::disM.SssI (blue lines), the control driver lines expressing only pFIL:LhG4 (red lines), the F2-Mss(-) plants carrying no transgenes (orange lines) and the F3-Mss(-) plants carrying only the pFIL::LhG4 transgene (green lines).The average methylation level of DMRs was determined by dividing the corresponding annotated regions into 100bp-bins.Regions located 2 kb upstream and 2 kb downstream of the CG hyperDMR are shown.